Ebola viruses cause acute, lethal hemorrhagic fevers for which no vaccines or treatments currently exist. Knowledge about the immune mechanisms mediating protection is limited. The membrane-anchored GP is the only viral protein known to be on the surfaces of virions and infected cells, and is presumed to be responsible for receptor binding and fusion of the virus with host cells. As a result, Ebola GP may be an important target of protective antibodies. However, the contribution of antibodies to Ebola GP in disease resistance is unclear. Negligible serum titers of neutralizing antibodies in convalescent patients, together with inconsistent results in achieving protection through experimental transfers of immune sera to animals (C. J. Peters and J. W. LeDuc, J. Infect. Dis. 179 (Suppl. 1), ix, 1999; V. V. Mikhailov et al., Vopr. Virusol. 39, 82, 1994) have led to suggestions that antibodies to Ebola GP cannot confer protection to Ebola virus (L. Xu et al., Nature Med. 4, 37, 1998).
The role of anti-GP antibodies in protection is further confounded by the observation that Ebola GP occurs in several forms. The transmembrane glycoprotein of Ebola viruses is unusual in that it is encoded in two open reading frames. Expression of GP occurs when the 2 reading frames are connected by transcriptional or translational editing (Sanchez et al., Proc. Natl. Acad. Sci. USA 93; 3602-3607, 1996; Volchkov et al., Virology 214, 421-430, 1995). The unedited GP mRNA produces a non-structural secreted glycoprotein (sGP) that is synthesized in large amounts early during the course of infection (Volchkov et al., 1995, supra; Sanchez et al., 1996, supra; Sanchez et al., J. Infect. Dis. 179 (suppl. 1, S164, 1999). Following editing, the virion-associated transmembrane glycoprotein is proteolytically processed into 2 disulfide-linked products (Sanchez et al., J. Virol. 72, 6442-6447, 1998). The amino-terminal product is referred to as GP1 (140 kDa) and the carboxy-terminal cleavage product is referred to as GP2 (26 kDa). GP1 and membrane-bound GP, covalently associate to form a monomer of the GP spike found on the surfaces of virions (V. E. Volchkov et al., Proc. Natl. Acad. Sci. U.S.A. 95, 5762, 1998; A. Sanchez et al., J. Virol. 72, 6442, 1998). GP1 is also released from infected cells in a soluble form (V. E. Volchkov. et al., Virology 245, 110, 1998). sGP and GP1 are identical in their first 295 N-terminal amino acids, whereas the remaining 69 C-terminal amino acids of sGP and 206 amino acids of GP1 are encoded by different reading frames. It has been suggested that secreted GP1 or sGP may effectively bind antibodies that might otherwise be protective (Sanchez et al., 1996, supra; Volchkov et al. 1998, supra).
Ebola virus GP is a type I transmembrane glycoprotein. Comparisons of the predicted amino acid sequences for the GPs of the different Ebola virus strains show conservation of amino acids in the amino-terminal and carboxy-terminal regions with a highly variable region in the middle of the protein (Feldmann et al., Virus Res. 24: 1-19, 1992). The GP of Ebola viruses are highly glycosylated and contain both N-linked and O-linked carbohydrates that contribute up to 50% of the molecular weight of the protein. Most of the glycosylation sites are found in the central variable region of GP.
Other studies have also demonstrated limited efficacy of passively transferred polyclonal antibodies in protection against Ebola challenge (Mikhailov et al, 1994, Voprosi Virusologii, 39, 82-84; Jahrling et al., 1996, Arch Virol, 11S, 135-140; Jahrling et al., 1999, J Infect Dis, 179 (Suppl 1), S224-234; Kudoyarova-Zubavichene et al., 1999, J Infect Dis, 179(Suppl 1), S218-223). However, it is difficult to determine the effective therapeutic dose of antibodies in different preparations of polyclonal antibodies. In addition, it is not known if monoclonal antibodies (MAbs) recognizing single epitopes on the Ebola GP are able to effectively neutralize or protect against Ebola virus in vivo.
This application describes protective GP-specific MAbs. The antibodies are classified into five groups based on competitive binding assays. Individual MAbs in these five groups were protective against Ebola challenge when administered prophylactically or therapeutically. Three of the epitopes bound by protective MAbs are linear sequences on GP1 whereas the other two are conformational epitopes shared between GP1 and sGP. Ten out of 14 MAbs identified in these five competition groups protected BALB/c mice from a lethal challenge with mouse-adapted Ebola Zaire virus when 100 ug of purified MAb was administered 24 hours before challenge (please see Table 3 in Examples below). Similar results were observed in a second mouse strain (C57BL/6). Protection from Ebola challenge decreased when the MAb dose was lowered to 50 or 25 ug (Please see Table 3 and Table 5 in Examples below). For the most effective MAbs, the amount required for protection was within an achievable human therapeutic dose of 3-5 mg/kg.
Some of the MAbs were effective even when administered up to 2 days after challenge (please see Table 3 in Examples below), after significant viral replication had occurred (M. Bray et al., J. Infect. Dis. 178, 651, 1998). None of the MAbs were protective when 100 ug was administered 3 days after challenge, when there are high viral titers (Bray et al., 1998, supra) and possibly irreversible damage of cells and organs.
The ability of the MAbs to inhibit plaque formation by Ebola virus, a standard assay of virus neutralization, did not always predict their protective efficacy. None of the protective MAbs inhibited plaque formation in the absence of complement (please see Table 6 in the Examples below).
Therefore, it is an object of the present invention to provide monoclonal antibodies which protect against Ebola virus and bind to epitopes on the Ebola virus GP. Such antibodies are, for instance, produced by any one of the cell lines deposited under the Budapest Treaty at American Type Culture Collection, Manassas, Va. on Jul. 20, 1999, EGP 13F6-1-2, assigned accession no. PTA-373, EGP6D3-1-1 assigned accession no. PTA-374, EGP 13-C6-1-1 assigned accession no. PTA-375, EGP 6D8-1-2 assigned accession no. PTA-376 and EGP 12B5-1-1 deposited on Jul. 29, 1999 and assigned accession no. PTA-436 (Table 1).
It is another object of the invention to provide for antibodies that are functionally equivalent to the antibodies listed above. These functionally equivalent antibodies substantially share at least one major functional property with an antibody listed above and herein described comprising: binding specificity to Ebola GP, protection against Ebola challenge when administered prophylactically or therapeutically, competition for same binding site on Ebola GP. The antibodies can be of any class such as IgG, IgM, or IgA or any subclass such as IgG1, IgG2a, and other subclasses known in the art. Further, the antibodies can be produced by any method, such as phage display, or produced in any organism or cell line, including bacteria, insect, mammal or other type of cell or cell line which produces antibodies with desired characteristics, such as humanized antibodies. The antibodies can also be formed by combining an Fab portion and a Fc region from different species.
The monoclonal antibodies of the present invention described below recognize epitopes on Ebola GP (SEQ ID NO: 1 and 2 describe the DNA and amino acid sequence, respectively, of Ebola GP used as an immunogen). Three epitopes are within the sequence extending from 389 to 493 and defined as: HNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLI (SEQ ID NO:3). More specifically, the cell line EGP 13F6-1-2 produces a monoclonal antibody 13F6 which recognizes and binds to an amino acid sequence of GP corresponding to a region extending from 401 to 417 (SEQ ID NO:4), recognizing an epitope within this region corresponding to Glu-Gln-His-His-Arg-Arg-Thr-Asp-Asn (SEQ ID NO:5). The cell line EGP 6D8-1-2 produces a monoclonal antibody 6D8 which recognizes and binds to an amino acid sequence of GP corresponding to a region extending from 389 to 405 (SEQ ID NO:6), recognizing an epitope within this region corresponding to Val-Tyr-Lys-Leu-Asp-Ile-Ser-Glu-Ala (SEQ ID NO:7). The cell line EGP 12B5-1-1 produces a monoclonal antibody 12B5 which recognizes and binds to an amino acid sequence of GP corresponding to a region extending from 477 to 493 (SEQ ID NO:8), recognizing an epitope within this region corresponding to Leu-Ile-Thr-Asn-Thr-Ile-Ala-Gly-Val (SEQ ID NO:9). The antibodies produced by cell lines EGP 13C6-1-1, 13C6, and EGP 6D3-1-1, 6D3, recognize conformational epitopes in GP sequence that may comprise discontinuous Ebola virus amino acids that are conserved between Zaire and Ivory Coast viruses and found in the 295 amino terminus of the protein (SEQ ID NO:10).
It is another object of the present invention to provide for mixtures of antibodies according to the present invention, as well as to methods of using individual antibodies, or mixtures thereof for the prevention and/or therapeutic treatment of Ebola infections in vitro and in vivo, and/or for improved detection of Ebola infections.
It is yet another object of the present invention to treat or prevent Ebola virus infection by administering a therapeutically or prophylactically effective amount of one antibody of the present invention or a mixture of antibodies of the present invention to a subject in need of such treatment.
It is another object of the present invention to provide passive vaccines for treating or preventing Ebola virus infections comprising a therapeutically or prophylactically effective amount of the antibodies of the present invention which protect against Ebola virus, in combination with a pharmaceutically acceptable carrier or excipient.
It is yet another object of the present invention to provide a method for diagnosis of Ebola virus infection by assaying for the presence of Ebola in a sample using the antibodies of the present invention.
It is still another object of the present invention to provide novel immunoprobes and test kits for detection of Ebola virus infection comprising antibodies according to the present invention. For immunoprobes, the antibodies are directly or indirectly attached to a suitable reporter molecule, e.g., and enzyme or a radionuclide. The test kit includes a container holding one or more antibodies according to the present invention and instructions for using the antibodies for the purpose of binding to Ebola virus to form an immunological complex and detecting the formation of the immunological complex such that presence or absence of the immunological complex correlates with presence or absence of Ebola virus.
It is another object of the present invention to provide anti-idiotypic antibodies raised against one of the present monoclonal antibodies for use as a vaccine to elicit an active anti-GP response.
It is yet another object of the present invention to provide antigenic epitopes as a component of a Ebola virus vaccine. The epitopes described above comprising SEQ ID NO:3-10, or conservative changes thereof which are still recognized by the antibodies, are useful for actively immunizing a host to elicit production of protective antibodies against Ebola.